RESUMO
Airway smooth muscle (ASM) cells express GABA A receptors (GABAARs), and previous reports have demonstrated that GABAAR activators relax ASM. However, given the activity of GABAARs in central nervous system inhibitory neurotransmission, concern exists that these activators may lead to undesirable sedation. MIDD0301 is a novel imidazobenzodiazepine and positive allosteric modulator of the GABAAR with limited brain distribution, thus eliminating the potential for sedation. Here, we demonstrate that MIDD0301 relaxes histamine-contracted guinea pig ( P < 0.05, n = 6-9) and human ( P < 0.05, n = 6-10) tracheal smooth muscle ex vivo in organ bath experiments, dilates mouse peripheral airways ex vivo in precision-cut lung-slice experiments ( P < 0.001, n = 16 airways from three mice), and alleviates bronchoconstriction in vivo in mice, as assessed by the forced-oscillation technique ( P < 0.05, n = 6 mice). Only trace concentrations of the compound were detected in the brains of mice after inhalation of nebulized 5 mM MIDD0301. Given its favorable pharmacokinetic properties and demonstrated ability to relax ASM in a number of clinically relevant experimental paradigms, MIDD0301 is a promising drug candidate for bronchoconstrictive diseases, such as asthma.
Assuntos
Asma/tratamento farmacológico , Barreira Hematoencefálica/efeitos dos fármacos , GABAérgicos/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Cobaias , Humanos , Ligantes , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Receptores de GABA-A/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismoRESUMO
We describe lead compound MIDD0301 for the oral treatment of asthma based on previously developed positive allosteric α5ß3γ2 selective GABAA receptor (GABAAR) ligands. MIDD0301 relaxed airway smooth muscle at single micromolar concentrations as demonstrated with ex vivo guinea pig tracheal rings. MIDD0301 also attenuated airway hyperresponsiveness (AHR) in an ovalbumin murine model of asthma by oral administration. Reduced numbers of eosinophils and macrophages were observed in mouse bronchoalveolar lavage fluid without changing mucous metaplasia. Importantly, lung cytokine expression of IL-17A, IL-4, and TNF-α were reduced for MIDD0301-treated mice without changing antiinflammatory cytokine IL-10 levels. Automated patch clamp confirmed amplification of GABA induced current mediated by α1-3,5ß3γ2 GABAARs in the presence of MIDD0301. Pharmacodynamically, transmembrane currents of ex vivo CD4+ T cells from asthmatic mice were potentiated by MIDD0301 in the presence of GABA. The number of CD4+ T cells observed in the lung of MIDD0301-treated mice were reduced by an oral treatment of 20 mg/kg b.i.d. for 5 days. A half-life of almost 14 h was demonstrated by pharmacokinetic studies (PK) with no adverse CNS effects when treated mice were subjected to sensorimotor studies using the rotarod. PK studies also confirmed very low brain distribution. In conclusion, MIDD0301 represents a safe and improved oral asthma drug candidate that relaxes airway smooth muscle and attenuates inflammation in the lung leading to a reduction of AHR at a dosage lower than earlier reported GABAAR ligands.
Assuntos
Asma/tratamento farmacológico , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Animais , Asma/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Líquido da Lavagem Broncoalveolar/química , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Constrição , Citocinas/metabolismo , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Feminino , Cobaias , Inflamação/metabolismo , Ligantes , Pulmão/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/metabolismo , Ovalbumina/metabolismo , Hipersensibilidade Respiratória/metabolismoRESUMO
We describe pharmacokinetic and pharmacodynamic properties of two novel oral drug candidates for asthma. Phenolic α4ß3γ2 GABAAR selective compound 1 and acidic α5ß3γ2 selective GABAAR positive allosteric modulator compound 2 relaxed airway smooth muscle ex vivo and attenuated airway hyperresponsiveness (AHR) in a murine model of asthma. Importantly, compound 2 relaxed acetylcholine contracted human tracheal airway smooth muscle strips. Oral treatment of compounds 1 and 2 decreased eosinophils in bronchoalveolar lavage fluid in ovalbumin sensitized and challenged mice, thus exhibiting anti-inflammatory properties. Additionally, compound 1 reduced the number of lung CD4+ T lymphocytes and directly modulated their transmembrane currents by acting on GABAARs. Excellent pharmacokinetic properties were observed, including long plasma half-life (up to 15 h), oral availability, and extremely low brain distribution. In conclusion, we report the selective targeting of GABAARs expressed outside the brain and demonstrate reduction of AHR and airway inflammation with two novel orally available GABAAR ligands.
Assuntos
Asma/patologia , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Eosinófilos/metabolismo , Citometria de Fluxo , Humanos , Pulmão , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/metabolismo , Receptores de GABA/metabolismo , Hipersensibilidade Respiratória/metabolismo , SuínosRESUMO
Recent studies have demonstrated that subtype-selective GABAA receptor modulators are able to relax precontracted human airway smooth muscle ex vivo and reduce airway hyper-responsiveness in mice upon aerosol administration. Our goal in this study was to investigate systemic administration of subtype-selective GABAA receptor modulators to alleviate bronchoconstriction in a mouse model of asthma. Expression of GABAA receptor subunits was identified in mouse lungs, and the effects of α4-subunit-selective GABAAR modulators, XHE-III-74EE and its metabolite XHE-III-74A, were investigated in a murine model of asthma (ovalbumin sensitized and challenged BALB/c mice). We observed that chronic treatment with XHE-III-74EE significantly reduced airway hyper-responsiveness. In addition, acute treatment with XHE-III-74A but not XHE-III-74EE decreased airway eosinophilia. Immune suppressive activity was also shown in activated human T-cells with a reduction in IL-2 expression and intracellular calcium concentrations [Ca(2+)]i in the presence of GABA or XHE-III-74A, whereas XHE-III-74EE showed only partial reduction of [Ca(2+)]i and no inhibition of IL-2 secretion. However, both compounds significantly relaxed precontracted tracheal rings ex vivo. Overall, we conclude that the systemic delivery of a α4-subunit-selective GABAAR modulator shows good potential for a novel asthma therapy; however, the pharmacokinetic properties of this class of drug candidates have to be improved to enable better beneficial systemic pharmacodynamic effects.
Assuntos
Asma/tratamento farmacológico , Benzodiazepinas/farmacologia , Receptores de GABA-A/metabolismo , Animais , Asma/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Cobaias , Humanos , Interleucina-2/metabolismo , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Ovalbumina/administração & dosagem , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Xenopus laevisRESUMO
A novel class of bivalent ligands targeting putative protease-activated receptor (PAR) heteromers has been prepared based upon reported antagonists for the subtypes PAR1 and PAR2. Modified versions of the PAR1 antagonist RWJ-58259 containing alkyne adapters were connected via cycloaddition reactions to azide-capped polyethylene glycol (PEG) spacers attached to imidazopyridazine-based PAR2 antagonists. Initial studies of the PAR1-PAR2 antagonists indicated that they inhibited G alpha q-mediated calcium mobilization in endothelial and cancer cells driven by both PAR1 and PAR2 agonists. Compounds of this novel class hold promise for the prevention of restenosis, cancer cell metastasis, and other proliferative disorders.
RESUMO
We report a 28-day repeat dose immunotoxicity evaluation of investigational drug MIDD0301, a novel oral asthma drug candidate that targets gamma amino butyric acid type A receptors (GABAA R) in the lung. The study design employed oral administration of mice twice daily throughout the study period with 100 mg/kg MIDD0301 mixed in peanut butter. Compound dosing did not reveal signs of general toxicity as determined by animal weight, organ weight or haematology. Peanut butter plus test drug (in addition to ad libitum standard rodent chow) did not affect weight gain in the adult mice, in contrast to weight loss in 5 mg/kg prednisone-treated mice. Spleen and thymus weights were unchanged in MIDD0301-treated mice, but prednisone significantly reduced the weight of those organs over the 28-day dosing. Similarly, no differences in spleen or thymus histology were observed following MIDD0301 treatment, but prednisone treatment induced morphological changes in the spleen. The number of small intestine Peyer's patches was not affected by MIDD0301 treatment, an important factor for orally administered drugs. Circulating lymphocyte, monocyte and granulocyte numbers were unchanged in the MIDD0301-treated animals, whereas differential lymphocyte numbers were reduced in prednisone-treated animals. MIDD0301 treatment did not alter IgG antibody responses to dinitrophenyl following dinitrophenyl-keyhole limpet haemocyanin immunization, indicating that systemic humoral immune function was not affected. Taken together, these studies show that repeated daily administration of MIDD0301 is safe and not associated with adverse immunotoxicological effects in mice.
Assuntos
Anti-Inflamatórios/administração & dosagem , Asma/tratamento farmacológico , Azepinas/administração & dosagem , Drogas em Investigação/administração & dosagem , Agonistas de Receptores de GABA-A/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Imidazóis/administração & dosagem , Tolerância Imunológica/efeitos dos fármacos , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Animais , Anti-Inflamatórios/efeitos adversos , Asma/sangue , Asma/imunologia , Azepinas/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/efeitos adversos , Feminino , Agonistas de Receptores de GABA-A/efeitos adversos , Hemocianinas/administração & dosagem , Hemocianinas/imunologia , Compostos Heterocíclicos com 3 Anéis/efeitos adversos , Humanos , Imidazóis/farmacologia , Contagem de Leucócitos , Masculino , Camundongos , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Redução de PesoRESUMO
Altered gamma-aminobutyric acid (GABA) function is consistently reported in psychiatric disorders, normal aging, and neurodegenerative disorders and reduced function of GABA interneurons is associated with both mood and cognitive symptoms. Benzodiazepines (BZ) have broad anxiolytic, but also sedative, anticonvulsant and amnesic effects, due to nonspecific GABA-A receptor (GABAA-R) targeting. Varying the profile of activity of BZs at GABAA-Rs is predicted to uncover additional therapeutic potential. We synthesized four novel imidazobenzodiazepine (IBZD) amide ligands and tested them for positive allosteric modulation at multiple α-GABAA-R (α-positive allosteric modulators), pharmacokinetic properties, as well as anxiolytic and antidepressant activities in adult mice. Efficacy at reversing stress-induced or age-related working memory deficits was assessed using a spontaneous alternation task. Diazepam (DZP) was used as a control. Three ligands (GL-II-73, GL-II-74, and GL-II-75) demonstrated adequate brain penetration and showed predictive anxiolytic and antidepressant efficacies. GL-II-73 and GL-II-75 significantly reversed stress-induced and age-related working memory deficits. In contrast, DZP displayed anxiolytic but no antidepressant effects or effects on working memory. We demonstrate distinct profiles of anxiolytic, antidepressant, and/or pro-cognitive activities of newly designed IBZD amide ligands, suggesting novel therapeutic potential for IBZD derivatives in depression and aging.
RESUMO
It is unclear whether GABAA receptors (GABAARs) that contain the α3-subunit are substantially involved in the anxiolytic effects of benzodiazepines (BDZs). In the present study, we tested YT-III-31, a newer BDZ ligand with functional preference for α3ßγ2 GABAARs, in two paradigms of unconditioned anxiety, the open field and elevated plus maze in rats. The effective dose of YT-III-31 (2â¯mg/kg) displayed a clear anxiolytic-like profile, unhampered by sedative action, in both tests. At a higher dose (10â¯mg/kg), YT-III-31 induced ataxia in the rotarod and sedation in spontaneous locomotor activity test. The latter effect was preventable by flumazenil and ßCCt, the non-selective and α1ßγ2 GABAAR affinity-selective antagonist, respectively, demonstrating that sedative properties of YT-III-31, when attained, are mediated by the α1γ2 site. To elucidate the receptor substrate of subtle behavioral differences between YT-III-31 and diazepam, we approximated in vivo receptor potentiation for both ligands, based on estimated unbound concentrations in rat brains. Far different from diazepam, YT-III-31 has significantly lower affinity for the α1γ2 over other BDZ-sensitive sites, and at lower doses (1-2â¯mg/kg) was devoid of potentiation at α1ßγ2 GABAARs. The approximation approach revealed a modest selectivity of YT-III-31 for α3γ2- in comparison to α2γ2 and α5γ2 binding sites, suggesting that its anxiolytic-like activity may not necessarily or predominantly reflect potentiation at α3ßγ2 GABAARs. Nonetheless, as the anxiolytic effects are achievable at a dose devoid of any sedative potential, and having favorable safety (cytotoxicity) and metabolic stability profile, YT-III-31 represents a valuable candidate for further translational research.
Assuntos
GABAérgicos/farmacologia , Receptores de GABA-A/metabolismo , Animais , Ansiolíticos/farmacologia , Ansiedade/tratamento farmacológico , Ansiedade/metabolismo , Benzodiazepinas/farmacologia , Sítios de Ligação , Ligação Competitiva , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Hipnóticos e Sedativos/farmacologia , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Ratos WistarRESUMO
A series of novel imidazobenzodiazepine analogs of the lead chiral ligand SH-053-2'F-S-CH3 (2), an α2/α3/α5 (Bz)GABA (A)ergic receptor subtype selective ligand, which reversed PCP-induced prepulse inhibition (PPI) of acoustic startle, were synthesized. These chiral (S)-CH3 ligands are targeted for the treatment of schizophrenia and depression. These new ligands were designed by modifying the liable ester functionality in 2 to improve the metabolic stability, cytotoxicity, and activity as compared to 2. Based on the data to date, the most promising ligands are the N-cyclopropyl amide GL-I-55 (8c) and the methyl bioisostere GL-I-65 (9a). The in vitro metabolic stability, cytotoxicity and in vivo locomotor effects are described in this report. Based on these results, 8c and 9a are the most promising for further in vivo pharmacology.
RESUMO
Recent reports indicate that α6ß2/3γ2 GABAAR selective ligands may be important for the treatment of trigeminal activation-related pain and neuropsychiatric disorders with sensori-motor gating deficits. Based on 3 functionally α6ß2/3γ2 GABAAR selective pyrazoloquinolinones, 42 novel analogs were synthesized, and their in vitro metabolic stability and cytotoxicity as well as their in vivo pharmacokinetics, basic behavioral pharmacology, and effects on locomotion were investigated. Incorporation of deuterium into the methoxy substituents of the ligands increased their duration of action via improved metabolic stability and bioavailability, while their selectivity for the GABAAR α6 subtype was retained. 8b was identified as the lead compound with a substantially improved pharmacokinetic profile. The ligands allosterically modulated diazepam insensitive α6ß2/3γ2 GABAARs and were functionally silent at diazepam sensitive α1ß2/3γ2 GABAARs, thus no sedation was detected. In addition, these analogs were not cytotoxic, which render them interesting candidates for treatment of CNS disorders mediated by GABAAR α6ß2/3γ2 subtypes.
Assuntos
Antagonistas GABAérgicos/síntese química , Antagonistas GABAérgicos/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Ansiolíticos/síntese química , Ansiolíticos/farmacologia , Comportamento Animal/efeitos dos fármacos , Disponibilidade Biológica , Deutério , Desenho de Fármacos , Feminino , Antagonistas GABAérgicos/farmacocinética , Células HEK293 , Humanos , Hipnóticos e Sedativos/farmacologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microssomos Hepáticos , Atividade Motora/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Ratos , Ratos Wistar , Especificidade por SubstratoRESUMO
γ-Aminobutyric acid type A (GABAA) receptors are located in spinal nociceptive circuits where they modulate the transmission of pain sensory signals from the periphery to higher centers. Benzodiazepine-type drugs bind to GABAA receptors containing α1, α2, α3, and α5 subunits (α1GABAA, α2GABAA, α3GABAA and α5GABAA receptors, respectively) through which they inhibit the transmission of these signals. In the present study we describe the novel benzodiazepine site positive allosteric modulator modulator methyl 8-ethynyl-6-(pyridin-2-yl)-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxylate (MP-III-024). MP-III-024 displayed preference for α2GABAA and α3GABAA receptors relative to α1GABAA and α5GABAA receptors as well as an improved metabolic profile relative to subtype-selective positive modulators that are available currently. Administration of MP-III-024 resulted in a dose- and time-dependent reversal of mechanical hyperalgesia. On locomotor activity and schedule-controlled responding, MP-III-024 was ineffective across the doses tested. These data provide further evidence that α2GABAA and α3GABAA receptors play an important role in the antihyperalgesic effects and may not be involved in some of the undesired effects of benzodiazepine-like drugs. Further, these findings suggest that MP-III-024 is a suitable research tool for investigating the role of α2GABAA and α3GABAA receptors in the behavioral properties of benzodiazepine-like drugs in mice.
Assuntos
Benzodiazepinas/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Hiperalgesia/tratamento farmacológico , Animais , Diazepam/farmacologia , Relação Dose-Resposta a Droga , Imidazóis/farmacologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de GABA-A/metabolismo , Ácido gama-AminobutíricoRESUMO
We describe the synthesis of analogs of XHE-III-74, a selective α4ß3γ2 GABAAR ligand, shown to relax airway smooth muscle ex vivo and reduce airway hyperresponsiveness in a murine asthma model. To improve properties of this compound as an asthma therapeutic, a series of analogs with a deuterated methoxy group in place of methoxy group at C-8 position was evaluated for isotope effects in preclinical assays; including microsomal stability, cytotoxicity, and sensorimotor impairment. The deuterated compounds were equally or more metabolically stable than the corresponding non-deuterated analogs and increased sensorimotor impairment was observed for some deuterated compounds. Thioesters were more cytotoxic in comparison to other carboxylic acid derivatives of this compound series. The most promising compound 16 identified from the in vitro screens also strongly inhibited smooth muscle constriction in ex vivo guinea pig tracheal rings. Smooth muscle relaxation, determined by reduction of airway hyperresponsiveness with a murine ovalbumin sensitized and challenged model, showed that 16 was efficacious at low methacholine concentrations. However, this effect was limited due to suboptimal pharmacokinetics of 16. Based on these findings, further analogs of XHE-III-74 will be investigated to improve in vivo metabolic stability while retaining the efficacy at lung tissues involved in asthma pathology.